Human circulating influenza-CD4+ ICOS1+IL-21+ T cells expand after vaccination, exert helper function, and predict antibody responses.

Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4(+) T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21(+) CD4(+) T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21(+) CD4(+) T cells can be measured in human blood, accumulate in the CXCR5(-)ICOS1(+) population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1(+)IL-21(+) CD4(+) T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5(-)ICOS1(+) CD4(+) T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1- and IL-21-dependent manner. We propose that the expansion of antigen-specific ICOS1(+)IL-21(+) CD4(+) T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.

Influenza vaccine immunology.

Studying the spread of influenza in human populations and protection by influenza vaccines provides important insights into immunity against influenza. The 2009 H1N1 pandemic has taught the most recent lessons. Neutralizing and receptor-blocking antibodies against hemagglutinin are the primary means of protection from the spread of pandemic and seasonal strains. Anti-neuraminidase antibodies seem to play a secondary role. More broadly cross-reactive forms of immunity may lessen disease severity but are insufficient to prevent epidemic spread. Priming by prior exposure to related influenza strains through infection or immunization permits rapid, potent antibody responses to immunization. Priming is of greater importance to the design of immunization strategies than the immunologically fascinating phenomenon of dominant recall responses to previously encountered strains (original antigenic sin). Comparisons between non-adjuvanted inactivated vaccines and live attenuated vaccines demonstrate that both can protect, with some advantage of live attenuated vaccines in children and some advantage of inactivated vaccines in those with multiple prior exposures to influenza antigens. The addition of oil-in-water emulsion adjuvants to inactivated vaccines provides enhanced functional antibody titers, greater breadth of antibody cross-reactivity, and antigen dose sparing. The MF59 adjuvant broadens the distribution of B-cell epitopes recognized on HA and NA following immunization.

Impact of preexisting memory to seasonal A/H1N1 influenza virus on the immune response following vaccination against avian A/H5N1 virus.

Cross-protection against divergent strains of influenza virus is an objective of various vaccination approaches. B cells cross-neutralizing several influenza A heterosubtypes have been isolated from cultured human memory B cells (MBCs) and plasmablasts early after influenza vaccination or infection. However, a systematic assessment of the frequency of MBCs and plasmablasts in the blood of healthy individuals is lacking. Here, we show that under resting conditions about 45% of human adults never vaccinated nor exposed to avian A/H5N1 influenza have detectable circulating MBCs cross-reacting with H5N1. This proportion rises to 63.3% among subjects with a large pool of MBCs specific for seasonal H1N1 (i.e. frequency ≥1% of total IgG MBCs). Moreover, subjects with high baseline frequencies of H1N1-specific MBCs had an expansion of H5N1-specific MBCs producing H5-neutralizing antibodies already after the first dose of an MF59-adjuvanted H5N1 vaccine. These results suggest that H1N1-specific MBCs contain a subset of cells cross-reacting to H5. We propose that a proportion of human adults have a pool of H5/H1 cross-reactive MBCs that contribute to the rapid rise of the antibody response to divergent influenza strains. This may have implications on vaccination strategies aimed at counteracting future influenza pandemics.

Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4(+) T cells broadly reactive with drifted H5. The CD4(+) response was dominated by IL-2(+) IFN-gamma(-) IL-13(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine.

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-microg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999-2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

NKT-cell help to B lymphocytes can occur independently of cognate interaction.

CD4(+) T (Th)-cell help to B lymphocytes requires cognate interaction and CD40 engagement. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that recognize alphagalactosylceramide (alphaGalCer) presented by CD1d, and can help B-cell responses. We asked whether alphaGalCer-activated iNKT cells help B lymphocytes through cognate interaction, or indirectly, via enhancement of Th-B-cell interaction. After immunization with protein Ags and alphaGalCer, antibody titers were assessed in wild-type or splenectomized mice, and in bone marrow radiation chimeras lacking CD1d or CD40 expression on B lymphocytes, or expressing CD1d or MHC II disjointly on antigen-presenting cells (APCs). We find that alphaGalCer-dependent enhancement of B-cell response (1) can occur when B cells do not express CD1d but express CD40; (2) requires that iNKT and Th cells interact with the same APCs that coexpress both CD1d and MHC-II; and (3) takes place without spleen. These findings demonstrate alphaGalCer-induced help for antibody responses can occur without cognate iNKT/B-cell interaction, and suggest this help entails activation of APCs by iNKT cells, which in turn activate Th cells and their helper functions for B cells. Thus, the alphaGalCer-induced help recapitulates the function of classical adjuvants that stimulate the innate immune system to support adaptive immune responses.

CD1d-restricted help to B cells by human invariant natural killer T lymphocytes.

Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen alpha-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of alpha-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4-CD8-), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.

Generating memory with vaccination.

The goal of vaccination is to induce long-lasting protective immune memory. Although most vaccines induce good memory responses, the type of memory induced by different vaccines may be considerably different. In addition, memory responses to the same vaccine may be influenced by age, environmental and genetic factors. Results emerging from detailed and integrated profiling of immune-responses to natural infection or vaccination suggest that the type and duration of immune memory are largely determined by the magnitude and complexity of innate immune signals that imprint the acquired immune primary responses. Here we summarize results obtained from analyzing human immune memory responses to different types of vaccines. We will also discuss how extending clinical investigation to events occurring early after vaccination can help identify early predictive markers of protective memory and thus contribute to faster development of better and safer vaccines.

Dissecting the immune response to MF59-adjuvanted and nonadjuvanted seasonal influenza vaccines in children less than three years of age.

INTRODUCTION: Annual seasonal influenza epidemics are particularly dangerous for the very young, the elderly and chronically ill individuals, in whom infection can cause severe morbidity, hospitalization and death. Existing, nonadjuvanted influenza vaccines exhibit a suboptimal immunogenicity and efficacy in immunologically naive subjects such as young children. METHODS: This phase II, randomized clinical trial was conducted to evaluate the antibody and cell-mediated responses to a trivalent influenza vaccine administered without adjuvant (TIV) or adjuvanted with MF59 (ATIV) in previously nonvaccinated children less than 3 years of age. RESULTS: The MF59-adjuvanted vaccine was well tolerated, and induced higher titers of hemagglutination inhibition antibodies able to recognize strains different from the one used in the vaccine (heterovariant) than TIV. The presence of the adjuvant MF59 induced a larger expansion of vaccine-specific CD4 T cells. Interestingly, the adjuvant MF59 did not modify the cytokine profile of the elicited T cells, characterized by the production of IL-2 and TNF-α, and did not bias the response toward either Th1 or Th2. The advantage of ATIV over TIV was more pronounced for the virus strains that had not circulated in the years that preceded this study and for the heterovariant strains. CONCLUSION: These data highlight the relevant role played by the oil-in-water adjuvant MF59 in enhancing the immunogenicity of inactivated influenza vaccines in immunologically naive individuals.

Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.

Superior immunogenicity of seasonal influenza vaccines containing full dose of MF59 (®) adjuvant: results from a dose-finding clinical trial in older adults.

BACKGROUND: MF59-adjuvanted influenza vaccines have superior immunogenicity in older adults compared with non-adjuvanted vaccines. We assessed whether changing formulation (i.e., increasing H3N2 antigen or decreasing the quantity of adjuvant) of the licensed, MF59-adjuvanted trivalent influenza subunit vaccine Fluad (®) (Novartis Vaccines and Diagnostics) improves the risk-benefit profile in vaccinees aged ≥ 65 years. RESULTS: A significant dose-response relationship was observed between antibody levels and MF59 dose; full dose formulations elicited the strongest immune responses, meeting immunogenicity licensure criteria by Day 8. Doubling H3N2 antigen content did not increase the response to this antigen. Increased frequency of circulating CD4+ T-cells specific for vaccine antigens were detected by Day 8; magnitude and functional profile of the CD4+ T-cell response was comparable across the different vaccination groups. Mild to moderate solicited local reactions were more common with vaccines formulated with higher doses of MF59 (®) , but there were no MF59- or antigen dose-related increase in the frequency of solicited systemic reactions or unsolicited adverse events and serious adverse events. METHODS: We report on 357 subjects who received one of eight intramuscular vaccine formulations. Hemagglutination-inhibiting antibodies were assayed on Days 1, 8 and 22; magnitude and functional profile of CD4+ T-cell responses to vaccine antigens were assessed in subsets. Solicited adverse reactions were reported via diary cards for seven days after vaccination and spontaneous adverse events were monitored throughout the study. CONCLUSION: This study confirms that the current formulation is the optimal one for MF59-adjuvanted influenza vaccine for use in older adults.

Lack of interference with immunogenicity of a chimeric alphavirus replicon particle-based influenza vaccine by preexisting antivector immunity.

Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens.

One dose of an MF59-adjuvanted pandemic A/H1N1 vaccine recruits pre-existing immune memory and induces the rapid rise of neutralizing antibodies.

Protective antibody responses to a single dose of 2009 pandemic vaccines have been observed in the majority of healthy subjects aged more than 3 years. These findings suggest that immune memory lymphocytes primed by previous exposure to seasonal influenza antigens are recruited in the response to A/H1N1 pandemic vaccines and allow rapid seroconversion. However, a clear dissection of the immune memory components favoring a fast response to pandemic vaccination is still lacking. Here we report the results from a clinical study where antibody, CD4+ T cell, plasmablast and memory B cell responses to one dose of an MF59-adjuvanted A/H1N1 pandemic vaccine were analyzed in healthy adults. While confirming the rapid appearance of antibodies neutralizing the A/H1N1 pandemic virus, we show here that the response is dominated by IgG-switched antibodies already in the first week after vaccination. In addition, we found that vaccination induces the rapid expansion of pre-existing CD4+ T cells and IgG-memory B lymphocytes cross-reactive to seasonal and pandemic A/H1N1 antigens. These data shed light on the different components of the immune response to the 2009 H1N1 pandemic influenza vaccination and may have implications in the design of vaccination strategies against future influenza pandemics.

Comparison of a limiting dilution assay and ELISpot for detection of memory B-cells before and after immunisation with a protein-polysaccharide conjugate vaccine in children.

In the present study, the limiting dilution assay (LDA) and the ELISpot are compared in their ability to detect serogroup C meningococcal (MenC)-specific memory B-cells in peripheral blood of 12month old children, after 3-dose priming with serogroup C meningococcal conjugate vaccine (MenCV) in infancy. At 12 months of age, MenC-memory B-cells were detected by ELISpot in 61% of children and in 5% by LDA. In contrast, carrier-specific memory B-cells were measurable in 36% of children by LDA and 78% by ELISpot. One month after a booster dose of MenCV given at 12 months of age, MenC-specific memory B-cells were detected in 89% of children by ELISpot and 65% of children by LDA, while diphtheria toxoid-specific memory B-cells were detected in 88% of children by ELISpot and in 74% of children by LDA. Two statistical methods were applied to enumerate memory B-cells with the LDA assay; the Poisson method allowed determination of very low frequencies that could not be determined by the Reed and Muench method. These data are examples of alternative methods to assess long-term protection after protein-polysaccharide conjugate vaccines, through direct measurement of memory B-cells in peripheral blood shortly after immunisation.

Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses.

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

The acquired immune response to the mucosal adjuvant LTK63 imprints the mouse lung with a protective signature.

LTK63, a nontoxic mutant of Escherichia coli heat labile enterotoxin (LT), is a potent and safe mucosal adjuvant that has also been shown to confer generic protection to several respiratory pathogens. To understand the mechanisms of action underlying the LTK63 protective effect, we analyzed the molecular and cellular events triggered by its administration in vivo. We show here that LTK63 intrapulmonary administration induced in the mouse lung a specific gene expression signature characterized by the up-regulation of cell cycle genes, several host defense genes, chemokines, chemokine receptors, and immune cell-associated genes. Such a transcriptional profile reflected the activation of alveolar macrophages and the recruitment to the lung of T and B cells and innate immune cells such as granulocytes, NK, and dendritic cells. All of these events were T cell dependent and specific for LTK63 because they were absent in SCID and nude mice. Additionally, we showed that LTK63 induces a potent adaptive immune response against itself directed to the lung. We propose that acquired response to LTK63 is the driving force for the local recruitment of both adaptive and innate immune cells. Our data suggest that LTK63 acts as an airway infection mimic that establishes a generic protective environment limiting respiratory infection by innate immune mechanisms and by improving adaptive responses to invading pathogens.

Invariant NKT cells sustain specific B cell responses and memory.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as alpha-galactosylceramide (alphaGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and alphaGC develop antibody titers 1-2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with alphaGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4(+) T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.

Innate immune responses support adaptive immunity: NKT cells induce B cell activation.

Invariant NKT cells are a peculiar subset of T lymphocytes whose features, highly conserved both in the mouse and the human system, strongly recall those of other "innate lymphocytes". Following recognition of CD1d-presented glycosphingolipid antigens invariant NKT promptly release high amount of diverse cytokines concurring to the activation of the actors of both innate and acquired immune responses. For this reason, in recent years NKT cells have been the object of intensive study, aimed to understand their role in diverse patho-physiological conditions and to exploit the possibility to take advantage of their "adjuvant-like" activity in the formulation of new vaccines. As antibodies are an essential part of many immune responses, we focused our attention on invariant NKT-B cell interactions analyzing their influences on B cell activation and effector functions. The results of this study demonstrate that human invariant NKT cells can provide direct help for B cell proliferation and antibody production through CD1d-restricted mechanisms. Remarkably, help to B lymphocytes by invariant NKT cells is delivered also in the absence of exogenous antigen, suggesting the existence of an endogenous ligand presented by CD1d on B cells.

Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis.

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.